Intestinal organoids mimicking Necrotizing Enterocolitis: a model to investigate the modulatory effects of Extracellular Vesicles from different sources

Necrotizing enterocolitis (NEC) is a severe neonatal disease with high morbidity and mortality. Despite numerous studies, its pathogenesis remains unclear, making NEC a complex clinical challenge due to its variability. Many experimental models fall short in replicating the disease because they overlook patient heterogeneity, complicating treatment. Traditional animal models and tissue cultures are often too simplistic. However, ex vivo platforms using patient biopsies more accurately simulate disease processes, allowing for experimental manipulations. In this thesis, we developed a 3D model as a promising tool to study NEC cellular mechanisms and investigate therapeutic interventions using extracellular vesicles (EVs) from different sources. In preliminary experiments, Caco-2 cells were exposed to lipopolysaccharide (LPS) and hypoxia to mimic NEC conditions. This combination caused the greatest decrease in TEER and disrupted the tight junction, making it suitable for NEC modeling. Histological analysis of human NEC samples revealed typical features, from mucosal to full-thickness necrosis, along with erosions, ulcerations, and hemorrhagic findings. Additionally, we found a significant decrease in occludin, a key tight junction protein, indicating compromised barrier integrity in NEC-affected neonates. We recognized that a cell culture model was inadequate and we advanced to a more complex model capable of replicating key aspects of pathophysiology ex vivo. Utilizing established protocols from the literature, we developed intestinal organoids derived from patient biopsies to NEC and we positivly stainined the organoids for markers, indicating the complexity of the cellular population. We induced experimental NEC through LPS e and hypoxia,revealing a compromise in barrier function, a reduction in the stem cell niche, and an increase in inflammation, thereby confirming the suitability of this model for mimicking NEC. To identify the most effective EVs for reducing intestinal damage in NEC, a systematic review and meta-analysis of preclinical studies were conducted. The analysis consistently showed that EVs, regardless of source reduced NEC incidence and severity in different models. Among them, MEVs were particularly effective in decreasing disease severity. Based on these findings, we tested the efficacy of different EV sources in an intestinal organoid model derived from rat intestines. EVs were administered at three stages—preventive, onset, and treatment—to assess their impact on inflammation and cellular regeneration. EVs significantly improved intestinal barrier function by suppressing proinflammatory gene expression and promoting cellular differentiation. However, the best outcomes for preserving tight junction integrity and restoring stem cells were observed when EVs were administered either at the time of injury or 48 hours after. Our study also identified human milk-derived EVs (HEVs) as the most effective at reducing inflammation, repairing the intestinal barrier, and enhancing stem cell recovery and proliferation—crucial for tissue regeneration.